1. Field of the Invention
The present invention relates to a method of assaying immunologically reactive substances of clinical interest. It also relates to a device for implementing this method.
2. Description of the Prior Art
Immunologically reactive substances means chiefly all the antigens (including the haptens) and the antibodies (monoclonal or polyclonal) produced by cell fusion, or by natural or induced immunization.
Immunologically reactive substances of clinical interest means chiefly all the antigen or antibody molecules whose assay in a biological specimen of human or animal origin may be of interest in medical diagnostics, clinical research or the monitoring of a pathological process or of a therapy which is employed.
The immunological phenomenon of which use is made in these immunoassay methods is shown in FIG. 1.
The most widely employed assay methods based on the use of the curve in FIG. 1 are:
radial immunodiffusion, PA1 radioimmunology, immunoenzymology, immunofluorescence; and PA1 nephelometry. PA1 particle counting, PA1 opacimetry, also known as turbidimetry (sometimes carried out in the visible or the infrared, in most cases in the near infrared), PA1 laser nephelometry, and PA1 centrifugal analysis.
Furthermore, some attempts at quantitative versions of the "latex immunotest", namely the agglutination of synthetic microspheres, have been described over a number of years. Until now, these techniques, which have been employed widely and for a long time (J. M. Singer and C. M. Plotz, The Latex fixation test, American Journal of Medicine 21, 888 (1956)), because of their simplicity and their low cost, were only qualitative (strip) or semiquantitative (well) in their form. The qualitative (strip) or semiquantitative forms (strip--sheet--well) of the Latex immunotest continue, furthermore, to be widely employed in clinical diagnostics at the present time and are the subject of many patents and publications, especially in the field of virology and microbiology (R. F. Khabbaz, H. C. Standford et al, 1985, Measurement of amikacin in serum by a Latex agglutination inhibition test, Journal of Clinical Microbiology, 22: 699-701, U.S. Pat. No. 3,488,156, European Patent Application 186 946). In these screening or detection techniques, the antigen or antibody is fixed on synthetic microspheres and the absence or presence of agglutination by the corresponding antigen or antibody is assessed in a tube or in a well, visually or by turbidimetry.
The few quantitative attempts at a Latex immunotest which are proposed in the literature (for example J. P. Ripoll, A. M. Roch, G. A. Quash and J. Grange, 1980, Journal of Immunological Methods 33, 159-173, European Patent Applications 189 389 and 5978) involve substantially three stages:
Stage I: Stage of fixing the antigen or the antibody on a micrometer of synthetic polymer, between 0.01 micrometer and 5 micrometers in size. The fixing is performed by any known means, which are identical with those described in the strip techniques (for example U.S. Pat. No. 4,217,338).
Stage II: Stage of immunological agglutination of the synthetic microspheres carrying the antigen or the antibody. This stage II of the method, namely the immunological agglutination of the synthetic microspheres, should aim at two objectives:
(1) the maximum reduction in the nonspecific agglutination of the particles; nonspecific agglutination means the agglutination of the particles which is due to weak interactions (of the hydrogen bond, ionic or van der Waals type), between the microspheres or the proteins which are fixed on the latter,
(2) the maximum increase in specific (immunological) agglutination of the particles.
During this stage, substantially chemical or biochemical means are employed to stabilize the particles, in order to reduce nonspecific agglutination to a minimum.
During this stage, means are also employed in order to increase the specific agglutination of the particles, and consequently the accuracy of the assay.
To reduce the nonspecific agglutination of the particles, U.S. Pat. No. 4,329,152 proposes to stabilize them by fixing bovine albumin made electronegative at a pH which is substantially equal to 10. The highly basic pH imposed in this manner on the reactant and on the calibration ranges is incompatible with survival of the proteins, and makes it a reactant which is difficult to employ. Furthermore, the reliability of the method is reduced thereby, because buffer solutions at pH values as basic as this are highly unstable. Furthermore, the bovine albumin fixed by hydrophobic bonding separates progressively from the particles at pH 10, and progressively loses its stabilizing capacity.
Unfortunately, the chaotropic agents which are sometimes employed to reduce the nonspecific agglutination of the particles have the disadvantage of weakening or breaking the antigen-antibody bonds.
Other stabilizing methods which are highly complicated and which cannot be employed in an industrial reactant make use of mixtures of heavy and light particles (ex: European Patent 163 312) and of centrifuging operations.
In order to promote the specific agglutination of the particles, that is to say immunological agglutination, the methods described employ conventional agitation, at a constant temperature in a water bath at 37.degree. C. or 40.degree. C., for periods ranging from one half hour to one hour. Such periods of incubation at 37.degree. C., in addition to promoting the undesirable nonspecific agglutination of the particles, demand strict and painstaking stopwatch timing of the initial introduction of the reactant into each tube in the series, and of the process of interrupting the reaction in the series of tubes.
An increase in the specific (immunological) agglutination of the particles is sometimes practiced by adding to the reaction medium additives such as, for example, dextrose, known under the trademark "Dextran" or polyethylene glycol. The disadvantage of these substances is that frequently they are to a large extent hydrophobic, and therefore they attach themselves to the Fc fragments of the antibodies fixed on the microparticles, fragments which are themselves highly hydrophobic, thus producing a nonimmunological agglutination of the particles, and thereby inducing interferences and, ipso facto, false positive reactions.
Stage III: Stage of reading the result.
At the present time the techniques of reading the result of the agglutination in quantitative latex immunotests are:
Laser nephelometry measures the light scattered by aggregates of latex particles. This technique, described, for example, in the following article: J. Grange, A. M. Roch and G. A. Quash, 1977, Journal of Immunological Methods 18, 326-375, introduces the disadvantage of requiring a costly and sophisticated reading apparatus.
Particle counting, described, for example, in the following article: C. G. Magnusson, P. L. Masson, Journal of Allergy Clin. and Immunol. 70: 326, 1982, also requires a complex and costly apparatus, accessible to only a few laboratories.
Opacimetry in the visible, also called turbidimetry in the visible, measures the light transmitted by the particle suspension, that is to say the light which is neither absorbed by the particles nor scattered by the latter. The opacimetry or turbidimetry of the latex immunotests, whether qualitative or quantitative, must be performed at a wavelength which lies fairly close to the particle size. Validity of the turbidimetric measurement calls for highly dilute suspensions, in order to enable the desired screening effect to be seen and, as indicated in the Certificate of Addition no 78/28,250 to french patent no 77/25,049, in order to reduce the absorption of light by the particles to a minimum.
Depending on the particle size, turbidimetric (or opacimetric) measurements in the visible of the latex immunotests described in the literature (example: A. M. Bernard, R. R. Lauwerys, 1982, clinica Chemica Acta 119, 335-339) employ various wavelengths in the visible, for example 360, 400, 450 and, much more widely, between 600 and 750 nanometres. The precision of the turbidimetric results is mediocre because the particle suspensions must be very dilute in order, as already stated, to produce the desired screeen effect variations and in order to reduce the absorption by the latices to a minimum. Furthermore, in order to increase the precision of the result, reading cells in which the optical path is long, of the order of two to four centimeters, must be generally employed in these techniques.
In order to employ more concentrated latex suspensions and thus to increase the precision of the assays, and in order to be free from interference by the absorption of the latices in the visible or in the ultraviolet, French Patent FR-A-77/25,049 and its Certificate of Addition FR-A-78/28250 recommend infrared opacimetry for particle reading. In point of fact, latices no longer absorb light in the infrared. The disadvantage of this technique is that it calls for costly and sophisticated apparatus for infrared opacimetric reading.